Understanding the function of commonly mutated tumor suppressor genes such as PTEN/MMAC1 and p16INK4A in human cancer cells is critical to elucidating the biological basis of cancer. Tumor suppressor genes also represent attractive drug targets, since mutations in them are one signature characteristic that distinguishes cancer cells from normal cells. In his graduate and post-doctoral studies, Todd Waldman M.D., Ph.D. developed novel approaches for modelling these inactivating tumor suppressor gene mutations in human cultured cells. In this Temin Award application, the candidate outlines a career development plan enabling him to mature into a fully independent investigator studying the biology of tumor suppressor gene function at the Lombardi Cancer Center. During years 1 and 2 of the award, the candidate will benefit from the co-mentorship of Dr. Marc Lippman, Director of the Lombardi Cancer Center, and Dr. Bert Vogelstein, an expert in the identification and study of tumor suppressor genes. By year 3, Dr. Waldman will be a fully independent investigator. The broad goal of this application is to establish an independent laboratory studying the roles of commonly-mutated tumor suppressor genes in human cancer cells. First, the applicant plans to study the roles of the PTEN/MMAC1 and p16INK4A tumor suppressor genes in the pathogenesis of human cancer by employing novel somatic cell gene targeting approaches that he developed during prior study of the p53 tumor suppressor gene pathway. The second broad goal, pursued in collaboration with Dr. Michael Boyd at the National Cancer Institute Laboratory of Drug Discovery Research and Development, is to exploit such isogenic systems for the purpose of anticancer drug discovery. The specific aims described in this Howard Temin Award proposal are: Aim number 1 is to develop and study isogenic systems of human cultured cells that differ only in the presence or absence of either the PTEN/MMAC 1 or p16INK4A tumor suppressor genes. Aim number 2 is to exploit an existing isogenic system of human cultured cells differing only in the presence or absence of their p21WAF1/CIP1 genes to identify small molecule drugs that specifically kill cells with checkpoint deficiencies. Aim number 3 is to develop new technologies and approaches to simplify gene targeting in human cultured cells.